Microorganisms and processes for the production of isoprene

ABSTRACT

The present invention provides a novel biosynthetic pathway for the production of isoprene from 3-methyl-2-buten-1-ol or 2-methyl-3-buten-2-ol. Further embodiments provide non-naturally occurring microorganism that have been modified to produce isoprene from 3-methyl-2-buten-1-ol or 2-methyl-3-buten-2-ol and methods of producing isoprene using said microorganism.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/776,485, filed on Mar. 11, 2013 and U.S. Provisional Application No.61/688,514, filed on May 16, 2012. The entire teachings of the aboveapplications are incorporated herein by reference.

FIELD OF THE INVENTION

The present disclosure generally relates to the use of a non-naturallyoccurring microorganism for the production of isoprene. Morespecifically, the present disclosure relates to non-naturally occurringmicroorganisms that have been modified to express enzymes that enablethe production of isoprene from different alcohols, in particular3-methyl-2-buten-1-ol or 2-methyl-3-buten-2-ol.

BACKGROUND OF THE INVENTION

Currently, many high-value chemicals or fuels are typically manufacturedby thermochemical processes from hydrocarbons, including petroleum oiland natural gas. Also, high value chemicals may be produced as“by-products” during the processing of crude oil into usable fractions.For example, isoprene has typically been produced during the catalyticcracking of crude oil fractions. However, recently catalytic crackerusers have shifted their focus from crude oil to natural gas, resultingin a reduced source of the four and five carbon chain molecules that arefound in crude oil, but not natural gas.

Being a short-chain carbon molecule, isoprene is a useful startingmaterial for synthesizing a variety of chemicals. Isoprene may be usedas a monomer or co-monomer for the production of higher value polymers.Examples of chemicals that can be produced using isoprene includepolyisoprene, polybutylene, styrene-isoprene-styrene block co-polymers,and others. An example of an industry that uses isoprene is thesynthetic rubber industry.

Given the increasing demand, decreasing supply and the many uses ofisoprene, a new method of isoprene production is desired. Also, as theconcerns of energy security, increasing oil and natural gas prices, andglobal warming escalate, the chemical production industry is seekingways to replace chemicals made from non-renewable feedstocks withchemicals produced from renewable feedstocks using environmentallyfriendly practices.

The biological production of isoprene has been studied since the 1950s(Sharkey, T. D. 2009. The Future of Isoprene Research. Bull. Georg.Natl. Acad. Sci. 3: 106-113). Although many different organisms areknown to emit isoprene, so far the biochemical pathway for isopreneproduction has only been elucidated in a few plant species. In plants,it appears that isoprene is produced in the chloroplast or otherplastids from dimethylallyl diphosphate, also referred to herein asdimethylallyl pyrophosphate (DMAPP), in a single step by isoprenesynthase, a nuclearly encoded enzyme that is routed to the plastid by aplastid targeting signal sequence. The isoprene synthases generally havea high Michaelis-Menten constant (K_(m)), typically 1 millimolar orhigher, and thus require high concentrations of dimethylallyldiphosphate to function efficiently.

Although microbes that naturally produce isoprene are known in the art(Kuzma, J., Nemecek-Marshall, M., Pollock, W. H., and R. Fall. 1995.Bacteria produce the volatile hydrocarbon isoprene. Curr. Microbiol. 30:97-103; Wagner, W. P., Nemecek-Marshall, M., and R. Fall. 1999. Threedistinct phases of isoprene formation during growth and sporulation ofBacillus subtilis. J. Bact. 181: 4700-4703; Fall, R. and S. D. Copley.2000. Bacterial sources and sinks of isoprene, a reactive atmospherichydrocarbon. Env. Microbiol. 2: 123-130; Xue, J., and B. K. Ahring.2011. Enhancing isoprene production by the genetic modification of the1-deoxy-D-xylulose-5-phosphate pathway in Bacillus subtilis. Appl. Env.Microbiol. 77: 2399-2405), the mechanism of isoprene production isunknown and the levels of isoprene production are relatively low.Several non-naturally occurring microorganisms have been engineered toproduce isoprene, e.g., U.S. patent application Ser. No. 12/335,071,wherein isoprene production requires an isoprene synthase. For efficientfunction of isoprene synthase, high intracellular levels ofdimethylallyl diphosphate are required; however, high levels ofintracellular dimethylallyl diphosphate are also toxic to the cells,retarding growth and reducing the rates and yields of isopreneproduction (Martin, V. J. J., Pitera, D. J., Withers, S. T., Newman, J.D. and J. D. Keasling. 2003. Engineering a mevalonate pathway inEscherichia coli for production of terpenoids. Nature Biotech. 21:796-802; Withers, S. T., Gottlieb, S. S., Lieu, B., Newman, J. D., andJ. D. Keasling. 2007. Identification of isopentenol biosynthetic genesfrom Bacillus subtilis by a screening method based on isoprenoidprecursor toxicity. Appl. Env. Microbiol. 73: 6277-7283; Sivy, T. L.,Fall, R., and T. N. Rosentiel. 2011. Evidence of isoprenoid precursortoxicity in Bacillus subtilis. Biosci. Biotechnol. Biochem. 75:2376-2383). The problems associated with the direct chemical conversionof DMAPP to isoprene by isoprene synthases limits the potential for thebiological production of commercially relevant amounts of isoprene.

Thus, there is a need for microorganisms and processes for the moreefficient biological production of isoprene.

SUMMARY OF THE INVENTION

Embodiments of the present invention generally provide enzymes,non-naturally occurring microorganisms, and methods of producingisoprene.

Embodiments of the invention provide non-naturally occurring microbialorganisms, i.e., microorganisms that include a biosynthetic isoprenepathway. The microorganisms include an exogenous nucleic acid encodingan enzyme of the biosynthetic pathway. The enzyme is a2-methyl-3-buten-2-ol dehydratase, and the biosynthetic pathway isexpressed at a sufficient level to produce isoprene. The biosyntheticpathway may further comprise a 2-methyl-3-buten-2-ol isomerase. The2-methyl-3-buten-2-ol isomerase may be part of a bi-functional enzymethat also has the 2-methyl-3-buten-2-ol dehydratase activity. An exampleof such a bi-functional enzyme is a linalool dehydratase-isomerase. Themicroorganism may further comprise a 3-methyl-2-buten-1-ol synthase.

In another embodiment, a non-naturally occurring microorganismcomprising a biosynthetic isoprene pathway is provided, wherein themicroorganism comprises an exogenous nucleic acid encoding an enzyme ofthe biosynthetic isoprene pathway, 2-methyl-3-buten-2-ol dehydratase.The pathway further comprises a 2-methyl-3-buten-2-ol synthase, and thepathway is expressed at a sufficient level to produce isoprene.

In one embodiment, the present invention provides for a non-naturallyoccurring microorganism comprising at least one or more exogenousnucleic acids encoding one or more enzymes of an isoprene biosyntheticpathway, wherein the one or more enzymes of an isoprene biosyntheticpathway are expressed in sufficient amounts to produce isoprene, saidisoprene biosynthetic pathway comprising a 3-methyl-2-buten-1-olsynthase, a 2-methyl-3-buten-2-ol isomerase, and a 2-methyl-3-buten-2-oldehydratase.

In another embodiment, the present invention provides for anon-naturally occurring microorganism comprising at least one or moreexogenous nucleic acids encoding one or more enzymes of an isoprenebiosynthetic pathway, wherein the one or more enzymes of an isoprenebiosynthetic pathway are expressed in sufficient amounts to produceisoprene, said isoprene biosynthetic pathway comprising a2-methyl-3-buten-2-ol synthase and a 2-methyl-3-buten-2-ol dehydratase.

In an additional embodiment, the present invention provides for a methodof producing isoprene, the method comprising the steps of culturing anon-naturally occurring microbial organism comprising at least one ormore exogenous nucleic acids encoding one or more enzymes of an isoprenebiosynthetic pathway, wherein the one or more enzymes of an isoprenebiosynthetic pathway are expressed in sufficient amounts to produceisoprene, in a suitable culture medium containing a carbon source underconditions such that the non-naturally occurring microorganism convertsat least a part of the carbon source to isoprene, and recovering theisoprene.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the manner in which the above recited features of the presentdisclosure can be understood in detail, a more particular description ofthe disclosure, briefly summarized above, may be had by reference toembodiments, some of which are illustrated in the appended drawings. Itis to be noted, however, that the appended drawings illustrate onlytypical embodiments of this invention and are therefore not to beconsidered limiting of its scope, for the invention may admit to otherequally effective embodiments.

FIG. 1 shows an isoprene biosynthetic pathway comprising a3-methyl-2-buten-1-ol synthase, a 2-methyl-3-buten-2-ol isomerase, and a2-methyl-3-buten-2-ol dehydratase.

FIG. 2 shows an isoprene biosynthetic pathway comprising a2-methyl-3-buten-2-ol synthase and a 2-methyl-3-buten-2-ol dehydratase.

FIG. 3 shows an E coli-codon-optimized nucleic acid sequence (SEQ ID NO:1), including an artificial ribosome binding site and an amino-terminal6-histidine epitope tag, for the linalool dehydratase isomerase ofCastellaniella defragrans strain 65Phen.

FIG. 4 shows an E coli-codon-optimized nucleic acid sequence (SEQ ID NO:2), including an artificial ribosome binding site and an amino-terminal6-histidine epitope tag, for strawberry alcohol acyltransferase.

FIG. 5 shows a gas chromatogram of a 1 ml sample of the headspace of a20-ml vial containing 1 mM 3-methyl-2-buten-1-ol dissolved in LuriaBertani broth. Peak 1 is 3-methyl-2-buten-1-ol, with a retention time of3.96 minutes.

FIG. 6 shows a gas chromatogram of a 1 ml sample of the headspace of a20 ml vial containing E. coli strain BL21 harboring plasmid pJ404-LDIcultured overnight on 1 mM 3-methyl-2-buten-1-ol in Luria Bertani brothsupplemented with 100 μg/ml ampicillin. Peak 1 is 3-methyl-2-buten-1-ol,with a retention time of 3.96 minutes. Peak 2 is 2-methyl-3-buten-2-olwith a retention time of 2.96 minutes. Peak 3 is isoprene, with aretention time of 2.49 minutes.

FIG. 7 shows a gas chromatogram of a 1 ml sample of the headspace of a20 ml containing 1 mM 2-methyl-3-buten-2-ol dissolved in Luria Bertanibroth. Peak 1 is 2-methyl-3-buten-2-ol with a retention time of 2.96minutes.

FIG. 8 shows a gas chromatogram of a 1 ml sample of the headspace of a20-milliliter vial containing E. coli strain BL21 harboring plasmidpJ404-LDI cultured overnight on 1 mM 2-methyl-3-buten-2-ol in LuriaBertani broth supplemented with 100 μg/ml ampicillin. Peak 1 is2-methyl-3-buten-2-ol with a retention time of 2.96 minutes. Peak 2 isisoprene with a retention time of 2.49 minutes.

FIG. 9 shows the results of GC/MS analysis of authentic isoprene.

FIG. 10 shows the results of GC/MS analysis of the peak at 2.49 minutesfrom Example 1, verifying the identity of the peak as isoprene.

FIG. 11 shows an E coli-codon-optimized nucleic acid sequence (SEQ IDNO: 3), including an artificial ribosome binding site, for the Bacillussubtilis yhfR gene.

FIG. 12 shows an E coli-codon-optimized nucleotide sequence (SEQ ID NO:4), including artificial binding sites and restriction endonucleasesites for subcloning, for a synthetic operon encoding FaNES 1 fromstrawberry and idi from H. pluvialis.

FIG. 13 shows a gas chromatogram of a 1 ml sample of the headspace of a20 ml vial containing 1 mM linalool dissolved in Luria Bertani broth.Peak 1 is linalool, with a retention time of 8.8 minutes.

FIG. 14 shows a gas chromatogram of a 1 ml sample of the headspace of a20 ml vial containing 1 mM 2-methyl-3-buten-2-ol dissolved in LuriaBertani broth under the same column conditions used to detect linalool.Peak 1 is 2-methyl-3-buten-2-ol, with a retention time of 4.8 minutes.

FIG. 15 shows a gas chromatogram of a 1 ml sample of headspace of a 20ml vial containing E. coli BL21 harboring plasmid pJ401-NES1-idicultured for 24 hours in Luria Bertani broth supplemented with 50 μg/mlkanamycin and 100 μM IPTG. Peak 1 is linalool, with a retention time of8.8 minutes. Peak 2 is 2-methyl-3-buten-2-ol, with a retention time of4.8 minutes. The peak at 4.75 minutes has been identified as 2-butanone.

FIG. 16 shows a gas chromatogram of a 1 ml sample of headspace of a 20ml vial containing E. coli BL21 harboring plasmid pJ404-SAAT culturedfor 24 hours in Luria Bertani broth supplemented with 100 μg/mlampicillin and 100 μM IPTG. Peaks corresponding to linalool and2-methyl-3-buten-2-ol are absent. The peak at 4.75 minutes has beenidentified as 2-butanone.

FIG. 17 shows the results of GC/MS analysis of authentic linalool.

FIG. 18 shows the results of GC/MS analysis of the peak at 8.8 minutesfrom FIG. 15, verifying the identity of the peak as linalool.

FIG. 19 shows the results of GC/MS analysis of authentic2-methyl-3-buten-2-ol.

FIG. 20 shows the results of GC/MS analysis of the peak at 4.9 minutesfrom FIG. 15, verifying the identity of the peak as2-methyl-3-buten-2-ol.

FIG. 21 shows the DNA sequence (SEQ ID NO: 5) of plasmid pGA31R-mcs.

FIG. 22 shows the DNA sequence (SEQ ID NO: 6) of plasmid pGS31R-mcs.

FIG. 23 shows the E coli-codon-optimized sequence (SEQ ID NO: 7) of themvaE and mvaS genes of Enterococcus faecalis ATCC 700802, includingincorporated ribosome binding sites and flanking restrictionendonuclease sites used in subsequent cloning steps.

FIG. 24 shows the E coli-codon-optimized sequence (SEQ ID NO: 8) of thesynthetic operon encoding the mevalonate kinase gene ofMethanocaldococcus jannaschi, the phosphomevalonate kinase gene ofEnterococcus faecalis ATCC 700802, the mevalonate diphosphatedecarboxylase gene of Saccharomyces cerevisiae S288C, and theisopentenyl diphosphate isomerase gene of E. coli MG1655, includingincorporated ribosome binding sites and flanking restrictionendonuclease sites used in subsequent cloning steps.

FIG. 25 shows a cloning strategy for the production of plasmid pGB 1026.

FIG. 26 shows a cloning strategy for the production of plasmid pGB1033.

FIG. 27 shows a cloning strategy for the production of plasmid pGB1036.

FIG. 28 shows a gas chromatogram of a 1 ml sample of headspace of a 20ml vial containing E. coli BL21 harboring plasmids pJ401-NEST-idi andpGB1036 cultured for 24 hours in Luria Bertani broth supplemented with50 μg/ml kanamycin, 20 μg/ml chloramphenicol, and 200 μg/mlanhydrotetracycline. Peak 1 is linalool, with a retention time of 8.8minutes. Peak 2 is 2-methyl-3-buten-2-ol, with a retention time of 4.8minutes. The peak at 4.75 minutes has been identified as 2-butanone.

DETAILED DESCRIPTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory only,and are not restrictive of the invention, as claimed. In thisapplication, the use of the singular includes the plural, the word “a”or “an” means “at least one”, and the use of “or” means “and/or”, unlessspecifically stated otherwise. Furthermore, the use of the term“including”, as well as other forms, such as “includes” and “included”,is not limiting. Also, terms such as “element” or “component” encompassboth elements or components comprising one unit and elements orcomponents that comprise more than one unit unless specifically statedotherwise.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including, but not limited to, patents, patent applications, articles,books, and treatises, are hereby expressly incorporated herein byreference in their entirety for any purpose. In the event that one ormore of the incorporated literature and similar materials defines a termin a manner that contradicts the definition of that term in thisapplication, this application controls.

As used herein, the term “non-naturally occurring” when used inreference to a microbial organism or microorganism of the invention isintended to mean that the microbial organism has at least one geneticalteration not normally found in a naturally occurring strain of thereferenced species, including wild-type strains of the referencedspecies. Genetic alterations include, for example, modificationsintroducing expressible nucleic acids encoding metabolic polypeptides,other nucleic acid additions, nucleic acid deletions and/or otherfunctional disruption of the microbial genetic material. Suchmodifications include, for example, coding regions and functionalfragments thereof, for heterologous, homologous or both heterologous andhomologous polypeptides for the referenced species. Additionalmodifications include, for example, non-coding regulatory regions inwhich the modifications alter expression of a gene or operon. Exemplarymetabolic polypeptides include enzymes or proteins within a glyceroldissimilation or isoprene biosynthetic pathway. As defined herein, an“isoprene biosynthetic pathway” comprises a pathway, e.g., a series ofone or more enzymes or activities involved in the production of isopreneby an organism, i.e., biologically, wherein one or more of those enzymesor activities is exogenous to the organism.

A metabolic modification refers to a biochemical reaction that isaltered from its naturally occurring state. Therefore, non-naturallyoccurring microorganisms can have genetic modifications to nucleic acidsencoding metabolic polypeptides or, functional fragments thereof.Exemplary metabolic modifications are disclosed herein.

As used herein, the term “isolated” when used in reference to amicrobial organism is intended to mean an organism that is substantiallyfree of at least one component as the referenced microbial organism isfound in nature. The term includes a microbial organism that is removedfrom some or all components as it is found in its natural environment.The term also includes a microbial organism that is removed from some orall components as the microbial organism is found in non-naturallyoccurring environments. Therefore, an isolated microbial organism ispartly or completely separated from other substances as it is found innature or as it is grown, stored or subsisted in non-naturally occurringenvironments. Specific examples of isolated microbial organisms includepartially pure microbes, substantially pure microbes and microbescultured in a medium that is non-naturally occurring.

As used herein, the terms “microbe,” “microbial,” “microbial organism”or “microorganism” is intended to mean any organism that exists as amicroscopic cell that is included within the domains of archaea,bacteria or eukarya. Therefore, the term is intended to encompassprokaryotic or eukaryotic cells or organisms having a microscopic sizeand includes bacteria, archaea and eubacteria of all species as well aseukaryotic microorganisms such as yeast and fungi. The term alsoincludes cell cultures of any species that can be cultured for theproduction of a biochemical.

“Exogenous” as it is used herein is intended to mean that the referencedmolecule or the referenced activity is introduced into the hostmicrobial organism. The molecule can be introduced, for example, byintroduction of an encoding nucleic acid into the host genetic materialsuch as by integration into a host chromosome or as non-chromosomalgenetic material such as a plasmid. Therefore, the term as it is used inreference to expression of an encoding nucleic acid refers tointroduction of the encoding nucleic acid in an expressible form intothe microbial organism. When used in reference to a biosyntheticactivity, the term refers to an activity that is introduced into thehost reference organism. The source can be, for example, a homologous orheterologous encoding nucleic acid that expresses the referencedactivity following introduction into the host microbial organism.Therefore, the term “endogenous” refers to a referenced molecule oractivity that is present in the host. Similarly, the term when used inreference to expression of an encoding nucleic acid refers to expressionof an encoding nucleic acid contained within the microbial organism. Theterm “heterologous” refers to a molecule or activity derived from asource other than the referenced species whereas “homologous” refers toa molecule or activity derived from the host microbial organism.Accordingly, exogenous expression of an encoding nucleic acid of theinvention can utilize either or both a heterologous or homologousencoding nucleic acid.

The non-naturally occurring microbial organisms of the invention cancontain stable genetic alterations, which refer to microorganisms thatcan be cultured for greater than five generations without loss of thealteration. Generally, stable genetic alterations include modificationsthat persist greater than 10 generations, particularly stablemodifications will persist more than about 25 generations, and moreparticularly, stable genetic modifications will be greater than 50generations, including indefinitely.

Those skilled in the art will understand that the genetic alterations,including metabolic modifications exemplified herein, are described withreference to a suitable host organism such as Escherichia coli and theircorresponding metabolic reactions or a suitable source organism fordesired genetic material such as genes for a desired metabolic pathway.However, given the complete genome sequencing of a wide variety oforganisms and the high level of skill in the area of genomics, thoseskilled in the art will readily be able to apply the teachings andguidance provided herein to essentially all other organisms. Forexample, the E. coli metabolic alterations exemplified herein canreadily be applied to other species by incorporating the same oranalogous encoding nucleic acid from species other than the referencedspecies. Such genetic alterations include, for example, geneticalterations of species homologs, in general, and in particular,orthologs, paralogs or non-orthologous gene displacements.

An ortholog is a gene or genes that are related by vertical descent andare responsible for substantially the same or identical functions indifferent organisms. For example, mouse epoxide hydrolase and humanepoxide hydrolase can be considered orthologs for the biologicalfunction of hydrolysis of epoxides. Genes are related by verticaldescent when, for example, they share sequence similarity of sufficientamount to indicate they are homologous, or related by evolution from acommon ancestor. Genes can also be considered orthologs if the proteinsthat they code for share three-dimensional structure but not necessarilysequence similarity, of a sufficient amount to indicate that they haveevolved from a common ancestor to the extent that the primary sequencesimilarity is not identifiable. Genes that are orthologous can encodeproteins with sequence similarity of about 25% to 100% amino acidsequence identity. Genes encoding proteins sharing an amino acidsimilarity less that 25% can also be considered to have arisen byvertical descent if their three-dimensional structure also showssimilarities. Members of the serine protease family of enzymes,including tissue plasminogen activator and elastase, are considered tohave arisen by vertical descent from a common ancestor.

Orthologs include genes or their encoded gene products that through, forexample, evolution, have diverged in structure or overall activity. Forexample, where one species encodes a gene product exhibiting twofunctions and where such functions have been separated into distinctgenes in a second species, the three genes and their correspondingproducts are considered to be orthologs. For the production of abiochemical product, those skilled in the art will understand that theorthologous gene harboring the metabolic activity to be introduced ordisrupted is to be chosen for construction of the non-naturallyoccurring microorganism. An example of orthologs exhibiting separableactivities is where distinct activities have been separated intodistinct gene products between two or more species or within a singlespecies. A specific example is the separation of elastase proteolysisand plasminogen proteolysis, two types of serine protease activity, intodistinct molecules as plasminogen activator and elastase. A secondexample is the separation of mycoplasma 5′-3′ exonuclease and DrosophilaDNA polymerase III activity. The DNA polymerase from the first speciescan be considered an ortholog to either or both of the exonuclease orthe polymerase from the second species and vice versa.

In contrast, paralogs are homologs related by structure or ancestry butwith different functions. These might arise by, for example, duplicationof a gene followed by evolutionary divergence to produce proteins withsimilar or common, but not identical functions. Paralogs can originateor derive from the same species or from a different species. Forexample, microsomal epoxide hydrolase (epoxide hydrolase I) and solubleepoxide hydrolase (epoxide hydrolase II) can be considered paralogsbecause they represent two distinct enzymes, co-evolved from a commonancestor, that catalyze distinct reactions and have distinct functionsin the same species. Paralogs are proteins from the same species withsignificant sequence similarity to each other suggesting that they arehomologous, or related through co-evolution from a common ancestor.Groups of paralogous protein families include HipA homologs, luciferasegenes, peptidases, and others.

A non-orthologous gene displacement is a non-orthologous gene from onespecies that can substitute for a referenced gene function in adifferent species. Substitution includes, for example, being able toperform substantially the same or a similar function in the species oforigin compared to the referenced function in the different species.Although generally, a non-orthologous gene displacement will beidentifiable as structurally related to a known gene encoding thereferenced function, less structurally related but functionally similargenes and their corresponding gene products nevertheless will still fallwithin the meaning of the term as it is used herein. Functionalsimilarity requires at least some structural similarity in the activesite or binding region of a non-orthologous gene product compared to agene encoding the function sought to be substituted. Therefore, examplesof non-orthologous genes include paralogs or unrelated genes.

Therefore, in identifying and constructing the non-naturally occurringmicrobial organisms of the invention having an isoprene biosyntheticpathway, those skilled in the art will understand with applying theteaching and guidance provided herein to a particular species that theidentification of metabolic modifications can include identification andinclusion or inactivation of orthologs. To the extent that paralogsand/or non-orthologous gene displacements are present in the referencedmicroorganism that encode an enzyme catalyzing a similar orsubstantially similar metabolic reaction, those skilled in the art alsocan utilize these evolutionally related genes. Orthologs, paralogs andnon-orthologous gene displacements can be determined by methods wellknown to those skilled in the art. As defined herein, enzymes or genesthat are described or claimed as being “derived from” an organisminclude any homologs, paralogs, non-orthologous gene displacements thathave substantially similar activity.

The methods and techniques utilized for culturing or generating themicroorganisms disclosed herein are known to the skilled worker trainedin microbiological and recombinant DNA techniques. Methods andtechniques for growing microorganisms (e.g., bacterial cells),transporting isolated DNA molecules into the host cell and isolating,cloning and sequencing isolated nucleic acid molecules, knocking outexpression of specific genes, etc., are examples of such techniques andmethods. These methods are described in many items of the standardliterature, which are incorporated herein in their entirety: “BasicMethods In Molecular Biology” (Davis, et al., eds. McGraw-HillProfessional, Columbus, Ohio, 1986); Miller, “Experiments in MolecularGenetics” (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1972); Miller, “A Short Course in Bacterial Genetics” (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1992); Singer andBerg, “Genes and Genomes” (University Science Books, Mill Valley,Calif., 1991); “Molecular Cloning: A Laboratory Manual,” 2^(nd) Ed.(Sambrook, et al., eds., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989); “Handbook of Molecular and Cellular Methodsin Biology and Medicine” (Kaufman, et al., eds., CRC Press, Boca Raton,Fla., 1995); “Methods in Plant Molecular Biology and Biotechnology”(Glick and Thompson, eds., CRC Press, Boca Raton, Fla., 1993); andSmith-Keary, “Molecular Genetics of Escherichia coli” (The GuilfordPress, New York, N.Y., 1989).

Although the direct conversion of dimethylallyl diphosphate to isopreneby isoprene synthase enzymes is known in the art, we have shown thatisoprene can also be produced from two different alcohols,3-methyl-2-buten-1-ol and 2-methyl-3-buten-2-ol, using linalooldehydratase-isomerase (see Example 1 and Example 2 and FIGS. 6 and 8),an enzyme isolated from Castellaniella defragrans strain 65Phen. Thelinalool dehydratase isomerase permits the development of novel isoprenebiosynthetic pathways of either two or three steps. In a two-stepisoprene biosynthetic pathway, dimethylallyl diphosphate is converted to2-methyl-3-buten-2-ol by an enzyme such as a 2-methyl-3-buten-2-olsynthase, followed by conversion of 2-methyl-3-buten-2-ol to isoprene.In a three-step isoprene biosynthetic pathway, dimethylallyl diphosphateis converted to 3-methyl-2-buten-1-ol by either a phosphatase or aterpene synthase capable of converting dimethylallyl diphosphate to3-methyl-2-buten-1-ol, 3-methyl-2-buten-1-ol is converted to2-methyl-3-buten-2-ol by a 2-methyl-3-buten-2-ol isomerase, and2-methyl-3-buten-2-ol is converted to isoprene by a2-methyl-3-buten-2-ol dehydratase. As demonstrated in Example 1 andExample 2, the Castellaniella defragrans linalool dehydratase-isomerasefunctions as both a 2-methyl-3-buten-2-ol isomerase and a2-methyl-3-buten-2-ol dehydratase.

Both the three-step isoprene biosynthetic pathway and the two-stepisoprene biosynthetic pathway are expressed at a sufficient level toproduce isoprene in detectable quantities. The isoprene may be detectedand characterized by gas chromatography/mass spectrometry, for example.

Three-Step Isoprene Biosynthetic Pathway

As used herein, enzyme names are defined as follows. A2-methyl-3-buten-2-ol dehydratase is an enzyme that catalyzes theconversion of 2-methyl-3-buten-2-ol to isoprene. A 2-methyl-3-buten-2-olsynthase is an enzyme that catalyzes the conversion of dimethylallyldiphosphate to 2-methyl-3-buten-2-ol. A 3-methyl-2-buten-1-ol synthaseor prenol synthase is an enzyme that catalyzes the conversion ofdimethylallyl diphosphate to 3-methyl-2-buten-1-ol also referred toherein as prenol. A 2-methyl-3-buten-2-ol isomerase is an enzyme thatcatalyzes the isomerization of 3-methyl-2-buten-1-ol to2-methyl-3-buten-2-ol.

In one embodiment of the present invention, a non-naturally occurringmicroorganism containing one or more exogenous genes encoding enzymes ofan isoprene biosynthetic pathway convert dimethylallyl diphosphate toisoprene in three steps (as used herein, “three-step isoprenebiosynthetic pathway,” FIG. 1). In a first step, dimethylallyldiphosphate is converted to 3-methyl-2-buten-1-ol by a3-methyl-2-buten-1-ol synthase. In a second step, 3-methyl-2-buten-1-olis converted to 2-methyl-3-buten-2-ol by a 2-methyl-3-buten-2-olisomerase. In a third step, 2-methyl-3-buten-2-ol is converted toisoprene by a 2-methyl-3-buten-2-ol dehydratase.

In a preferred embodiment of a naturally occurring microorganism for theconversion of dimethylallyl diphosphate to isoprene in three steps, thefirst step is catalyzed by a 3-methyl-2-buten-1-ol synthase and thesecond and third steps are catalyzed by a single, bi-functional enzymewith both 2-methyl-3-buten-2-ol isomerase and 2-methyl-3-buten-2-oldehydratase activities.

The conversion of dimethylallyl diphosphate to 3-methyl-2-buten-1-ol(prenol) may be catalyzed by a phosphatase. Examples of suchphosphatases include enzymes encoded by the Bacillus subtilis genes yqkG(nudF) and yhfR (Withers, S. T., Gottlieb, S. S., Lieu, B., Newman, J.D. and J. D. Keasling. 2007. Identification of isopentenol biosyntheticgenes from Bacillus subtilis by a screening method based on isoprenoidprecursor toxicity. Appl. Env. Microbiol. 73: 6277-6283), although otherknown phosphatases and coding sequences with predicted phosphataseactivity, for example the ytjC gene of E. coli, may be used. Table 1,below, provides examples of phosphatases for use in the conversion ofdimethylallyl diphosphate to prenol.

TABLE 1 Locus GenBank Accession No. Organism BAA12639 (YqkG) BAA12639Bacillus subtilis CAA74541 (YhfR) CAA74541 Bacillus subtilis subsp.subtilis Strain 168 GPMB_ECOLI P0A7A2 Escherichia coli K-12 CalfIntestine Alkaline Phosphatase Shrimp Alkaline Phosphatase

The conversion of dimethylallyl diphosphate to 3-methyl-2-buten-1-ol maybe catalyzed by a terpene synthase, e.g., a geraniol synthase orfarnesol synthase or mutants thereof, for example. Table 2, below,provides examples of terpene synthases for use in the conversion ofdimethylallyl diphosphate to 3-methyl-2-buten-1-ol.

TABLE 2 Locus GenBank Accession No. Organism Geraniol Synthase AAR11765AAR11765 Ocimum basilicum ABB30216 ABB30216 Perilla citriodora ABB30217ABB30217 Perilla citriodora ABB30218 ABB30218 Perilla frutescensCAE52821 CAE52821 Cinnamomum tenuipile Farnesol Synthase ACSS_MAIZEQ84ZW8 Zea mays ABJ16554 ABJ16554 Oryza sativa

3-methyl-2-buten-1-ol is isomerized to 2-methyl-3-buten-2-ol by a2-methyl-3-buten-2-ol isomerase. As used herein, a 2-methyl-3-buten-2-olisomerase is an enzyme that converts 3-methyl-2-buten-1-ol (prenol) to2-methyl-3-buten-2-ol in a reversible reaction. An example of such anenzyme is the linalool dehydratase-isomerase of Castellanielladefragrans strain 65Phen, GenBank accession number FR669447. This enzymecatalyzes the isomerization of 3-methyl-2-buten-1-ol to2-methyl-3-buten-2-ol and the dehydration of 2-methyl-3-buten-2-ol toisoprene (Example 1, below, and FIG. 6). Orthologs, paralogs andnon-orthologous gene displacements of linalool dehydratase-isomerase canbe determined by methods well known to those skilled in the art.

As used herein, a 2-methyl-3-buten-2-ol dehydratase is an enzyme thatconverts 2-methyl-3-buten-2-ol to isoprene. An example of such an enzymeis the linalool dehydratase-isomerase of Castellaniella defragransstrain 65Phen, GenBank accession number FR669447. This enzyme is capableof catalyzing the dehydration of 2-methyl-3-buten-2-ol to isoprene(Example 2, below, and FIG. 8). Orthologs, paralogs and non-orthologousgene displacements of linalool dehydratase-isomerase can be determinedby methods well known to those skilled in the art. For example,inspection of nucleic acid or amino acid sequences for two polypeptideswill reveal sequence identity and similarities between the comparedsequences. Based on such similarities, one skilled in the art candetermine if the similarity is sufficiently high to indicate theproteins are related through evolution from a common ancestor.Algorithms well known to those skilled in the art, such as Align, BLAST,Clustal W and others compare and determine a raw sequence similarity oridentity, and also determine the presence or significance of gaps in thesequence that can be assigned a weight or score. Such algorithms alsoare known in the art and are similarly applicable for determiningnucleotide sequence similarity or identity. Parameters for sufficientsimilarity to determine relatedness are computed based on well knownmethods for calculating statistical similarity, or the chance of findinga similar match in a random polypeptide, and the significance of thematch determined. A computer comparison of two or more sequences can, ifdesired, also be optimized visually by those skilled in the art. Relatedgene products or proteins can be expected to have a high similarity, forexample, 25% to 100% sequence identity. Proteins that are unrelated canhave an identity that is essentially the same as would be expected tooccur by chance, if a database of sufficient size is scanned (about 5%).Sequences between 5% and 24% may or may not represent sufficienthomology to conclude that the compared sequences are related. Additionalstatistical analysis to determine the significance of such matches giventhe size of the data set can be carried out to determine the relevanceof these sequences.

Exemplary parameters for determining relatedness of two or moresequences using the BLAST algorithm, for example, can be as set forthbelow. Briefly, amino acid sequence alignments can be performed usingBLASTP version 2.0.8 (Jan. 5, 1999) and the following parameters:Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50;expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignmentscan be performed using BLASTN version 2.0.6 (Sep. 16, 1998) and thefollowing parameters: Match: 1; mismatch: −2; gap open: 5; gapextension: 2; x_dropoff: 50; expect: 10.0; wordsize: 11; filter: off.Those skilled in the art will know what modifications can be made to theabove parameters to either increase or decrease the stringency of thecomparison, for example, and determine the relatedness of two or moresequences.

2-methyl-3-buten-2-ol dehydratase enzyme activity has also beenidentified in Aquincola tertiaricarbonis (Schuster, J., Schäfer, F.,Hübler, N., Brandt, A., Rosell, M., Härtig, C., Harms, h., Müller, R. H.and T. Rohwerder. 2012. Bacterial degradation of tert-amyl alcoholproceeds via hemiterpene 2-methyl-3-buten-2-ol by employing the tertiaryalcohol desaturase function of the Rieske nonheme mononuclear ironoxygenase MdpJ. J. Bact. 194: 972-981). The sequence of this2-methyl-3-buten-2-ol dehydratase has not been reported.

Two-Step Isoprene Biosynthetic Pathway

In another embodiment, a non-naturally occurring microorganismcontaining one or more exogenous genes encoding enzymes of an isoprenebiosynthetic pathway convert dimethylallyl diphosphate to isoprene intwo steps catalyzed by a 2-methyl-3-buten-2-ol synthase (MBO synthase)and a 2-methyl-3-buten-2-ol dehydratase (as used herein, “two-stepisoprene biosynthetic pathway,” FIG. 2).

As used herein, an example of a 2-methyl-3-buten-2-ol synthase is anaturally occurring polypeptide found in some plant plastids,particularly in the chloroplast, that converts dimethylallyl diphosphateto 2-methyl-3-buten-2-ol, and derivatives (mutants) of polypeptides thatnaturally convert dimethylallyl diphosphate to 2-methyl-3-buten-2-ol.MBO synthases are characterized, in part, by an amino-terminal plastidtargeting sequence that routes the polypeptide to the chloroplast. Upontranslocation into the chloroplast, the transit peptide may be cleavedfrom the polypeptide to yield a mature protein that is smaller inmolecular weight than the precursor protein. For overexpression of anexogenous MBO synthase in a microbial organism, it is preferable toexpress a truncated MBO synthase that approximates the mature form foundin nature, rather than the precursor form. Essentially, the sequenceencoding the transit peptide is removed from the MBO synthase codingsequence. While visual inspection may allow one skilled in the art toselect where to truncate the isoprene synthase coding sequence,computer-based algorithms such as ChloroP 1.1 can be used to helppredict which amino acids belong to the transit peptide (Emanuelsson,0., Nielsen, H., G. von Heijne. 1999. ChloroP, a neural network-basedmethod for predicting chloroplast transit peptides and their cleavagesites. Protein Sci. 8: 978-984). An example of an MBO synthase is foundin Pinus sabiniana, with the GenBank accession number AEB53064.1.

The conversion of dimethylallyl diphosphate to 2-methyl-3-buten-2-ol maybe catalyzed by a terpene synthase, e.g., a linalool synthase (e.g. E.C.No. 4.2.3.25 or 4.2.3.26) or nerolidol synthase or mutants thereof, forexample. Table 3, below, provides examples of terpene synthases for usein the conversion of dimethylallyl diphosphate to 2-methyl-3-buten-2-ol.

TABLE 3 Locus GenBank Accession No. Organism S-Linalool SynthaseLIS_CLABR Q96376 Clarkia breweri LINS_ARATH Q84UV0 Arabidopsis thalianaC0KWV3_9LAMI C0KWV3 Perilla setoyensis C0KWV5_PERFR C0KWV5 Perillafrutescens var. hirtella C0KWV7_PERFR C0KWV7 Perilla frutescens var.hirtella D4N3A0_9ERIC D4N3A0 Actinidia arguta D4N3A1_9ERIC D4N3A1Actinidia polygama R-Linalool Synthase LLOS1_ARTAN Q9SPN0 Artemesiaannua LLOS_OCIBA Q5SBP3 Ocimum basilicum LLOS5_ARTAN Q9SPN1 Artemesiaannua LLOS_MENAQ Q8H2B4 Mentha aquatica Q1XBU5_SOLLC Q1XBU5 Solanumlycopersicum (3S,6E)-Nerolidol Synthase Q5UB06_MEDTR Q5UB06 Medicagotrunculata F8TWD1_POPTR F8TWD1 Populus trichocarpa NES1_FRAVE P0CV96Fragaria vesca NES1_FRAAN P0CV94 Fragaria ananassa NES2_FRAAN P0CV95Fragaria ananassaOne example of the use of a terpene synthase to convert dimethylallyldiphosphate to 2-methyl-3-buten-2-ol is found in Example 3.

The conversion of 2-methyl-3-buten-2-ol to isoprene may be catalyzed bya 2-methyl-3-buten-2-ol dehydratase as described above. The2-methyl-3-buten-2-ol dehydratase may be a bi-functional enzyme withboth 2-methyl-3-buten-2-ol isomerase and 2-methyl-3-buten-2-oldehydratase activities, such as the linalool dehydratase-isomerasedescribed above, or the enzyme may encode only the 2-methyl-3-buten-2-oldehydratase activity without a 2-methyl-3-buten-2-ol isomerase activity.

In a preferred embodiment of the present invention, dimethylallyldiphosphate available for conversion to isoprene by either a two-stepisoprene biosynthetic pathway or a three-step isoprene biosyntheticpathway may be increased by overexpression of one or more endogenousgenes or expression of one or more exogenous genes encoding enzymes ofthe methylerythritol phosphate pathway: 1-deoxy-D-xylulose-5-phosphatesynthase, 1-deoxy-D-xylulose-5-phosphate reductoisomerase,4-diphosphocytidyl-2-C-methyl-D-erythritol synthase,4-diphosphocytidyl-2-C-methyl-D-erythritol kinase,2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase,1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase,dimethylallyl-diphosphate/isopentenyl-diphosphate:NAD(P)⁺oxidoreductase, or isopentenyl diphosphate isomerase. For example,expression of exogenous genes encoding 1-deoxy-D-xylulose-5-phosphatesynthase, 1-deoxy-D-xylulose-5-phosphate reductoisomerase, andisopentenyl diphosphate isomerase may result in increased levels ofdimethylallyl diphosphate, and when expressed in conjunction with eithera two-step isoprene biosynthetic pathway or a three-step isoprenebiosynthetic pathway, result in increased yields of isoprene.

In another preferred embodiment of the present invention, dimethylallyldiphosphate available for conversion to isoprene by either a two-stepisoprene biosynthetic pathway or a three-step isoprene biosyntheticpathway may be increased by expression of one or more exogenous genesencoding enzymes of the mevalonate pathway including, but not limitedto: acetyl-CoA acetyltransferase (also known as thiolase),3-hydroxy-3-methylglutaryl-CoA synthase, 3-hydroxy-3-methylglutaryl-CoAreductase, mevalonate kinase, phosphomevalonate kinase,diphosphomevalonate decarboxylase, and isopentenyl diphosphateisomerase. One example of overexpression of exogenous genes encoding themevalonate pathway is described in Example 4.

In an additional embodiment, the present invention provides for a methodof producing isoprene, the method comprising the steps of culturing anon-naturally occurring microbial organism comprising at least one ormore exogenous nucleic acids encoding one or more enzymes of an isoprenebiosynthetic pathway, wherein the one or more enzymes of an isoprenebiosynthetic pathway are expressed in sufficient amounts to produceisoprene, in a suitable culture medium containing a carbon source underconditions such that the non-naturally occurring microorganism convertsat least a part of the carbon source to isoprene, and recovering theisoprene. The carbon source may be or comprise glycerol, glucose,xylose, arabinose, or mixtures thereof; dimethylallyl diphosphate,3-methyl-2-buten-1-ol, or 2-methyl-3-buten-2-ol. Preferably, the carbonsource is or comprises glycerol, glucose or sugars derived fromcellulosic biomass processes. The isoprene may be recovered as describedin the examples below.

In the following examples of embodiments of the current invention, thecommon E. coli strain BL21 was used for the examples. BL21 (LifeTechnologies, Inc., Carlsbad, Calif.) cells were made electrocompetentand electroporated following the protocol from the MicroPulserElectroporation Apparatus Operating Instructions and Applications Guide(Bio-Rad catalog number 165-2100), except that LB without salt was usedto grow up the culture in making cells electrocompetent.

EXAMPLE 1 Microorganism for the Production of Isoprene from3-Methyl-2-Buten-1-ol

This working example shows the production of isoprene from3-methyl-2-buten-1-ol by a non-naturally occurring microorganismexpressing one or more exogenous genes of an isoprene biosyntheticpathway.

The plasmid pJ404-LDI was constructed by DNA2.0 (Menlo Park, Calif.)using the E coli-codon-optimized sequence (SEQ ID NO: 1) of the linalooldehydratase-isomerase (LDI) of Castellaniella defragrans strain 65Phen.The LDI coding sequence was codon-optimized for expression in E. coli,synthesized and inserted into the plasmid expression vectorpJexpress404. The resulting plasmid, pJ404-LDI, was electroporated intoE. coli BL21 electrocompetent cells.

Plasmid pJ404-SAAT was constructed by DNA2.0 (Menlo Park, Calif.) usingthe codon-optimized sequence (SEQ ID NO: 2) of the strawberry acyl-CoAtransferase (SAAT). The SAAT coding sequence was codon-optimized forexpression in E. coli, synthesized and inserted into the plasmidexpression vector pJexpress404. The resulting plasmid, pJ404-SAAT, waselectroporated into E. coli BL21 electrocompetent cells. pJ404-SAAT wasused as a negative control.

Transformants of BL21 harboring either pJ404-LDI or pJ404-SAAT wereselected on Luria-Bertani (LB)-agar plates (10 g/L yeast extract, 5 g/LBacto Tryptone, 10 g/L sodium chloride, 15 g/L Bacto Agar) containing100 μg/ml ampicillin.

A single colony of BL21 harboring pJ404-LDI or pJ404-SAAT from theLB-agar plates was used to inoculate 10 ml of LB broth (10 g/L yeastextract, 5 g/L Bacto Tryptone, 10 g/L sodium chloride) containing 100μg/ml ampicillin contained in 125-mL Erlenmeyer flasks. Flasks wereincubated for 16 hours at 37° C. in a rotary shaking incubator. After 16hours, the resulting cultures were diluted using fresh LB brothcontaining 100 μg/ml ampicillin to an optical density of 0.16 at 600 nm.50 ml of the diluted cultures were placed in 300-ml Erlenmeyer flasksand incubated at 37° C. in a rotary shaking incubator until the opticaldensity at 600 nm reached approximately 0.6, typically 90 minutes. 4 mlof the resulting cultures were then placed into 20 ml gas chromatographyheadspace vials. 3-methyl-2-buten-1-ol was added to a finalconcentration of 1 mM, IPTG (Isopropyl β-D-1-thiogalactopyranoside) wasadded to 0.1 mM, and the cultures were grown for an additional 16 hoursat 37° C. with shaking.

Isoprene was measured using headspace analysis on an Agilent 7890A GCequipped with a CTC-PAL autosampler and a FID. Headspace vials (20 ml)were incubated at 50° C. with agitation at 500 rpm for 2 minutes. Then 1ml of the headspace was removed using a heated headspace syringe at 50°C. and injected into the GC inlet (250° C., split of 20:1). Samples wereanalyzed using a FID detector set at 300° C., with a helium carrier gasflow rate of 2 ml/min through a DB-624 30 m×530 μm×3 μm column (J&WScientific), and an oven program of 85° C. for 5.25 minutes. Theisoprene concentration in samples was calculated from calibration curvesgenerated from isoprene calibration gas standards analyzed under thesame GC/FID method. The isoprene product was also confirmed by headspaceGC/MS using an Agilent 7890A GC equipped with a 5975C MSD and a CTC-PALautosampler. Headspace vials were incubated at 85° C. with agitation at600 rpm for 5 minutes. Then 1 ml of the headspace was removed using aheated headspace syringe at 85° C. and injected into the GC inlet (250°C., split of 25:1). The GC/MS method used helium as the carrier gas at 1ml/min through a HP-5MS 30 m×250 μm×0.25 μm column (J&W Scientific), anoven program of 35° C. for 4 minutes, then ramped 25° C./min to 150° C.,a MS source temperature of 230° C., and a quadrupole temperature of 150°C. The mass spectrometer was operated in scan mode from 25 to 160 massunits. The isoprene peak was identified by the NIST 11 MS Library, aswell as comparison against an authentic sample (135 ppm isoprene, 135ppm carbon dioxide in dry nitrogen gas, Matheson TRIGAS, Houston, Tex.).

3-methyl-2-buten-1-ol and 2-methyl-3-buten-2-ol were measured usingheadspace analysis on an Agilent 7890A GC equipped with a CTC-PALautosampler and a FID. Headspace vials (20 ml) were incubated at 85° C.with agitation at 600 rpm for 5 minutes. Then 1 ml of the headspace wasremoved using a heated headspace syringe at 85° C. and injected into theGC inlet (250° C., split of 25:1). Samples were analyzed using a FIDdetector set at 350° C., with a helium carrier gas flow rate of 3 ml/minthrough at DB-624 30 m×530 μm×3 μm column (J&W Scientific), and an ovenprogram of 90° C., then ramping 20° C./min to 230° C. for 3 minutes. The3-methyl-2-buten-1-ol and 2-methyl-3-buten-2-ol concentrations insamples were calculated from calibration curves generated from dilutedstandards of each compound analyzed under the same GC/FID method.

The results of this example are presented in FIG. 5 and FIG. 6. LB brothcontaining 1 mM 3-methyl-2-buten-1-ol without E. coli cells showed apeak at 3.96 minutes corresponding to 3-methyl-2-buten-1-ol (FIG. 5).Similarly, cultures containing 1 mM 3-methyl-2-buten-1-ol with BL21cells harboring pJ404-SAAT showed a peak at 3.96 minutes correspondingto 3-methyl-2-buten-1-ol, and an additional peak corresponding to thealdehyde 3-methyl-2-buten-1-al (prenal, data not shown). In contrast,cultures containing 1 mM 3-methyl-2-buten-1-ol with BL21 cells harboringpJ404-LDI converted 3-methyl-2-buten-1-ol to 2-methyl-3-buten-2-ol andisoprene, corresponding to peaks at 2.96 minutes and 2.49 minutes,respectively. This demonstrates that E. coli cells harboring pJ404-LDIisomerize 3-methyl-2-buten-1-ol to 2-methyl-3-buten-2-ol and dehydrate2-methyl-3-buten-2-ol to isoprene. FIG. 9 presents the GC/MS analysis ofan authentic isoprene sample; FIG. 10 presents the GC/MS analysis of thepeak with a 2.49 minute retention time, with the same fragmentationpattern as authentic isoprene shown in FIG. 9.

EXAMPLE 2 Microorganism for the Production of Isoprene from2-Methyl-3-Buten-2-ol

This working example shows the production of isoprene from2-methyl-3-buten-2-ol by a non-naturally occurring microorganismexpressing one or more exogenous genes of an isoprene biosyntheticpathway.

A single colony of BL21 harboring pJ404-LDI or pJ404-SAAT from LB-agarplates was used to inoculate 10 ml of LB broth (10 g/L yeast extract, 5g/L Bacto Tryptone, 10 g/L sodium chloride) containing 100 μg/mlampicillin contained in 125-mL Erlenmeyer flasks. Flasks were incubatedfor 16 hours at 37° C. in a rotary shaking incubator. After 16 hours,the cultures were diluted using fresh LB broth containing 100 μg/mlampicillin to an optical density of 0.16 at 600 nm. 50 ml of the dilutedcultures were placed in 300-mL Erlenmeyer flasks and incubated at 37° C.in a rotary shaking incubator until the optical density at 600 nmreached approximately 0.6, typically 90 minutes. 4 ml of the cultureswere then placed into 20-ml gas chromatography headspace vials.2-methyl-3-buten-2-ol was added to a final concentration of 1 mM. IPTG(Isopropyl β-D-1-thiogalactopyranoside) was added to a finalconcentration of 0.1 mM. Cultures containing 2-methyl-3-buten-2-ol weregrown for 16 hours at 37° C. with shaking.

Isoprene, 3-methyl-2-buten-1-ol and 2-methyl-3-buten-2-ol were measuredas above. The identity of the isoprene peak was also verified usingGC/MS, as described above in Example 1.

The results of this example are presented in FIG. 7 and FIG. 8. LB brothcontaining 1 mM 2-methyl-3-buten-2-ol with E. coli cells omitted showeda peak at 2.96 minutes corresponding to 2-methyl-3-buten-2-ol (FIG. 7).Similarly, cultures containing 1 mM 2-methyl-3-buten-2-ol and BL21 cellsharboring pJ404-SAAT showed a peak at 2.96 minutes corresponding to2-methyl-3-buten-2-ol (data not shown). In contrast, cultures containing1 mM 2-methyl-3-buten-2-ol and BL21 cells harboring pJ404-LDI converted2-methyl-3-buten-2-ol to isoprene, corresponding to the peak at 2.49minutes. This demonstrates that E. coli cells harboring pJ404-LDIdehydrate 2-methyl-3-buten-2-ol to isoprene.

EXAMPLE 3 FaNES1 Catalyzes Formation of 2-Methyl-3-Buten-2-ol fromDimethylallyl Diphosphate

This working example shows the production of 2-methyl-3-buten-2-ol fromdimethylallyl diphosphate by a non-naturally occurring microorganismexpressing an exogenous terpene synthase, the (3S,6E)-nerolidol synthaseof Fragaria ananassa.

The plasmid pJ401-NEST-idi was constructed by DNA2.0 (Menlo Park,Calif.) using the codon-optimized sequence of the (3S,6E)-nerolidolsynthase, FaNES1, of Fragaria ananassa (GenBank accession no. P0CV94;Aharoni, A., Giri, A. P., Verstappen, F. W. A., Bertea, C. M., Sevenier,R., Sun, Z., Jongsma, M. A., Schwab, W. and H. J. Bouwmeester. 2004.Gain and loss of fruit flavor compounds produced by wild and cultivatedstrawberry species. The Plant Cell 16: 3110-3131) and thecodon-optimized isopentenyl diphosphate isomerase gene, idi, of H.pluvialis. Both the FaNES1 and idi coding sequences were codon-optimizedfor expression in E. coli, synthesized and inserted into the plasmidexpression vector pJexpress401. The resulting plasmid, pJ401-NES1-idi,was electroporated into E. coli BL21 electrocompetent cells. Thecodon-optimized sequence (SEQ ID NO: 4), including artificial ribosomalbinding sites and flanking restriction endonucleases for subcloning, isprovided in FIG. 12.

The production of nerolidol, linalool and 2-methyl-3-buten-2-ol wasassayed as follows. A single colony of BL21 harboring plasmidpJ401-NES1-idi from an LB-agar plate was used to inoculate 10 ml of LBbroth (10 g/L yeast extract, 5 g/L Bacto Tryptone, 10 g/L sodiumchloride) containing 50 μg/ml kanamycin. Flasks were incubated for 16hours at 37° C. in a rotary shaking incubator. After 16 hours, thecultures were diluted using fresh LB broth containing 50 μg/ml kanamycinand 0.1 mM IPTG to yield an initial cell density at 600 nm of 0.4 to0.5. 4 mL of the diluted culture was placed in a 20 ml GC vial andincubated for 6 or 24 hours at 30° C. with shaking. At 6 or 24 hours,the headspace gas was analyzed by GC/MS-SIM.

Samples were analyzed by headspace GC/MS in Select Ion Mode (SIM) usingan Agilent 7890A GC equipped with a 5975C MSD and a CTC-PAL autosampler.Headspace vials (20 ml) were incubated at 85° C. with agitation at 600rpm for 5 minutes. Then 1 ml of the headspace was removed using a heatedheadspace syringe at 85° C. and injected into the GC inlet (250° C.,split of 25:1). Helium was used as the carrier gas at 1.5 ml/min througha VF-624MS 60 m×250 μm×1.4 μm column (J&W Scientific) and an ovenprogram of 90° C. for 1 minute, then ramped 25° C./min to 230° C. for 5min. The mass spectrometer was operated in SIM mode. The MS sourcetemperature was 230° C., the quadrupole temperature was 150° C., and thesolvent delay was 3.55 min. Concentrations of target analytes weredetermined from calibration curves of each analyte. Calibrationstandards for 2-methyl-3-buten-2-ol, 3-methyl-2-buten-1-ol,3-methyl-2-butenal, and linalool were prepared in 10 mL of deionizedwater at concentrations of 1, 10, and 100 ppm. The headspace for eachcalibration standard was analyzed using the same GC/MS-SIM method.Isoprene was calibrated from certified gas standards at 14, 135, and1375 ppm. Linear correlation coefficients for calibration curveswere >0.99 for all impurity components. FIG. 13 shows a gas chromatogramof authentic linalool acquired under the GC/MS-SIM conditions. FIG. 14shows a gas chromatogram of authentic 2-methyl-3-buten-2-ol acquiredunder the GC/MS-SIM conditions.

The results of this example are presented in FIG. 15. BL21 cellsharboring pJ401-NES1-idi produced 1.47 mg/L linalool, corresponding tothe peak at 8.8 minutes, and 0.05 mg/L 2-methyl-3-buten-2-ol,corresponding to the peak at 4.8 minutes. This demonstrates that E. colicells harboring pJ401-NEST-idi produce 2-methyl-3-buten-2-ol in additionto linalool. The peaks corresponding to linalool and2-methyl-3-buten-2-ol are absent from control cultures of BL21 harboringpJ404-SAAT grown under similar conditions (FIG. 16). The peaks wereidentified by the NIST 11 MS Library. The peaks for linalool (FIG. 17and FIG. 18) and 2-methyl-3-buten-2-ol (FIG. 19 and FIG. 20) were alsoidentified by comparison of retention times and ion fragmentationpatterns against authentic samples.

EXAMPLE 4 Overexpression of Mevalonate Pathway to improve2-methyl-3-buten-2-ol production by FaNES1

This working example shows the production of 2-methyl-3-buten-2-ol fromdimethylallyl diphosphate by a non-naturally occurring microorganismexpressing an exogenous terpene synthase, the (3S,6E)-nerolidol synthaseof Fragaria ananassa, can be enhanced by overexpression of aheterologous mevalonate pathway to increase the pool of dimethylallyldiphosphate available for conversion to 2-methyl-3-buten-2-ol.

The heterologous mevalonate pathway was constructed on a plasmid,pGB1036, as follows.

Plasmid pGA31R-MCS was constructed entirely by DNA synthesis, with thenucleotide sequence (SEQ ID NO: 5) presented in FIG. 21.

Plasmid pGS31R-MCS was constructed by replacing the p15A origin ofreplication on pGA31R-MCS with the low-copy pSC101 origin as anAvrII/SacI fragment using standard cloning techniques. The nucleotidesequence (SEQ ID NO: 6) is provided in FIG. 22.

Plasmid pJ248-mvaES was constructed using the codon-optimized sequence(SEQ ID NO: 7) of the mvaE and mvaS genes of Enterococcus faecalis ATCC700802 (the codon-optimized sequences of mvaE and mvaS are as presentedin FIG. 23). The mvaE and mvaS genes of Enterococcus faecalis ATCC700802 were codon-optimized for expression in E. coli, synthesized andinserted in the plasmid pJ248. Unique ribosomal binding sites wereincluded in front of each gene, along with flanking endonucleaserestriction sites for use in plasmid construction.

Plasmid pJ241-MK.PMK.MPD.IDI containing a codon-optimized syntheticoperon was constructed entirely by DNA synthesis, with the nucleotidesequence (SEQ ID NO: 8) presented in FIG. 24. The sequence of thesynthetic operon, codon-optimized for expression in E. coli, encodes themevalonate kinase gene of Methanocaldococcus jannaschi, thephosphomevalonate kinase of Enterococcus faecalis ATCC 700802, themevalonate diphosphate decarboxylase of Saccharomyces cerevisiae S288C,and the isopentenyl diphosphate isomerase gene of E. coli MG1655,including incorporated ribosomal binding sites and flanking restrictionendonuclease sites used in subsequent cloning steps.

Plasmid pGB 1008 was constructed by cloning the optimized mvaES genesfrom pJ248-mvaES into pGA31R-MCS as a KpnI/MluI DNA fragment usingstandard cloning techniques.

Plasmid pGB1026. The cloning strategy for pGB1026 is presented in FIG.25. Plasmid pGB1026 was constructed by inserting an approximately 3,000base pair PCR product encoding the pntAB genes of E. coli into the MluIsite of pGB 1008. The PCR product encoding the pntAB genes was amplifiedfrom genomic DNA of MG1655 using AccuPrime Pfx polymerase with thefollowing oligonucleotide primers:

Primer 1: (SEQ ID NO: 9) 5′- CCG TAA CTA AAC GCG AAG GGA ATA TCA TGC GAA TTG G -3′ Primer 2: (SEQ ID NO: 10)5′- CTA GAG ATC TAC GCG TCA GGG TTA CAG AGC  TTT C -3′

Primer 1 incorporates a ribosomal binding site in front of the startcodon of pntA. Primers 1 and 2 also include appropriatevector-overlapping 5′ sequences for use with the In-Fusion Advantage PCRCloning Kit (Clontech). The PCR product was gel-purified, as was pGB1008linearized with the restriction endonuclease MluI. Fragments weredirectionally joined together using the In-Fusion cloning kit and GC5competent cells, following the manufacturer's directions. Transformantswere screened, and the proper plasmid was identified through agarose gelelectrophoresis of restriction endonuclease-digested plasmid DNAs.

Plasmid pGB1033 was created through the following process, illustratedin FIG. 26. pGB1026 was digested with the restriction endonucleases NcoIand SphI; the resulting 8.3 kb fragment was gel-purified. A secondaliquot of pGB 1026 was digested with the restriction endonucleases MluIand SphI; the resulting 1.4 kb fragment was gel-purified. PlasmidpJ241-MK.PMK.MPD.IDI was digested with the restriction endonucleasesNcoI and MluI; the resulting 4.1 kb containing the synthetic operon wasgel-purified. The fragments were ligated together in a trimolecularligation reaction using the NEB Quick Ligation Kit (New England BioLabs)and transformed into GC5 competent cells. Transformants were screened,and the proper plasmid was identified through agarose gelelectrophoresis of restriction endonuclease-digested plasmid DNAs.

Plasmid pGB1036 was constructed by cloning the 2 operons from pGB1033,complete with promoters and terminators into pGS31R-MCS as a BamHI/AvrIIDNA fragment using standard cloning techniques, as illustrated in FIG.27. The fragments were ligated together using the NEB Quick Ligation Kit(New England BioLabs) and transformed into GC5 competent cells.Transformants were screened, and the proper plasmid was identifiedthrough agarose gel electrophoresis of restriction endonuclease-digestedplasmid DNAs.

Plasmids pGB1036 and pJ401-NEST-idi, were co-transformed byelectroporation into E. coli BL21 electrocompetent cells.

The production of nerolidol, linalool and 2-methyl-3-buten-2-ol wasassayed as follows. A single colony of BL21 harboring plasmidspJ401-NES1-idi and pGB1036 from an LB-agar plate was used to inoculate10 ml of LB broth (10 g/L yeast extract, 5 g/L Bacto Tryptone, 10 g/Lsodium chloride) containing 50 μg/ml kanamycin and 37 μg/mlchloramphenicol. Flasks were incubated for 16 hours at 37° C. in arotary shaking incubator. After 16 hours, the cultures were dilutedusing fresh LB broth containing 50 μg/ml kanamycin, 20 μg/mlchloramphenicol, 200 μg/ml ahydrotetracycline, and 0.1 mM IPTG to yieldan initial cell density at 600 nm of 0.4 to 0.5. 4 ml of the dilutedculture was placed in a 20 ml GC vial and incubated for 6 or 24 hours at30° C. with shaking. At 6 or 24 hours, the headspace gas was analyzed byGC/MS-SIM as described in Example 3.

The results of this example are presented in FIG. 28. BL21 cellsharboring pJ401-NEST-idi and pGB1036 produced approximately 4.05 mg/Llinalool, corresponding to the peak at 8.8 minutes, and 0.38 mg/L2-methyl-3-buten-2-ol, corresponding to the peak at 4.8 minutes. Thisdemonstrates that E. coli cells harboring pJ401-NEST-idi and pGB1036produce over 7 times more 2-methyl-3-buten-2-ol than cells harboringpJ401-NES1-idi by itself.

EXAMPLE 5 FaNES1 Mutations that Alter the Linalool to2-Methyl-3-Buten-2-ol Ratio

This working example shows that the introduction of mutations intoFaNES1 can alter the amount of linalool produced as compared to theamount of 2-methyl-3-buten-2-ol produced.

Site-directed mutagenesis or complete gene synthesis were used tointroduce specific amino acid substitutions into the wild-type FaNES1amino acid sequence. Table 4 presents the names of the mutant enzymesand the associated mutations. The amino acid numbering presented inTable 4 and this example correspond with the amino acid positions in thewild-type FaNES 1 enzyme as reported in GenBank accession no. P0CV94.

TABLE 4 Enzyme Name Introduced Mutation(s) NES1v2 I266F, S374F and I490FNES1#1 I266F NES1#2 S374F NES1#3 I490F NES1#4 G375D NES1#5 I266F andS374F NES1#6 I266F and 1490F NES1#7 S374F and I490F NES1#8 L413F NES1#9I490K NES1#10 I490Y

NESv2 was produced from plasmid pJ401-NES1v2-idi using thecodon-optimized sequence of the FaNES1 and the H. pluvialis idi genes.During construction, three amino acid mutations were introduced,converting the isoleucine at position 266 to phenylalanine (I266F), theserine at position 374 to phenylalanine (S374F), and the isoleucine atposition 490 to phenylalanine (I490F).

FaNES1 mutants NES1#1 through NES1#10 were created through standardsite-directed mutagenesis techniques using plasmid pJ401-NES1v2-idi as atemplate. The site-directed mutations were confirmed through DNAsequencing. Confirmed mutants were electroporated into E. coli BL21electrocompetent cells. The production of linalool and2-methyl-3-buten-2-ol for each individual mutant was assayed accordingto the methods described in Example 3, with a culture time of 6 hours at30° C. The results are presented in Table 5.

TABLE 5 2-methyl-3-buten-2-ol FaNES1 Variant Enzyme Linalool (mg/L)(mg/L) Wild-type 0.54 0.02 NESv2 — — NES#1 — — NES#2 — — NES#3 — — NES#4— — NES#5 — — NES#6 — — NES#7 — — NES#8 0.05 — NES#9 — — NES#10 — —

Since it had been shown (Example 4) that increasing the supply ofdimethylallyl diphosphate results in increased production of linalooland 2-methyl-3-buten-2-ol by wild-type FaNES1, a subset of the plasmidsencoding FaNES1 variants were co-transformed with pGB1036 into BL21electrocompetent cells. The ability of the variant enzymes to producelinalool and 2-methyl-3-buten-2-ol was assayed according to the methodspresented in Example 3, with a 24 hour incubation at 30° C. The resultsare presented in Table 6.

TABLE 6 FaNES1 Variant Enzyme* Lin* (mg/L) 232-MB* (mg/L) Wild-type 4.050.38 NES1#1 0.04 NES1#3 0.08 0.04 NES1#8 0.79 0.05 NES1#9 XX XX NES1#10XX XX *In vivo assay performed with enhanced dimethylallyl diphosphateconcentrations provided by pGB1036; Lin = Linalool; 232-MB =2-methyl-3-buten-2-ol.

EXAMPLE 6 Microorganism for the Production of Isoprene fromDimethylallyl Diphosphate

This example demonstrates how one may produce isoprene with anon-naturally occurring microorganism expressing a phosphatase, a2-methyl-3-buten-2-ol isomerase, and a 2-methyl-3-buten-2-oldehydratase.

The yhfR gene of Bacillus subtilis was codon-optimized for expression inE. coli, synthesized and inserted into the plasmid vector pJex404 toproduce pJex404-yhfR. The codon-optimized yhfR sequence (SEQ ID NO: 3),including a ribosome binding site, is presented in FIG. 11. The ribosomebinding site and yhfR coding sequence were amplified by polymerase chainreaction (PCR) using the following oligonucleotide primers:

(SEQ ID NO: 11) 5′- GGG CAA GTA ACT CGA TTA AAG AGG AGA AAATAT AAT GAC GGC AG -3′ (SEQ ID NO: 12)5′- GCC CTT GGG GCT CGA GTT ATT TGA TGA AAC CGC TCA GAT GG -3′

Plasmid pJ404-LDI was linearized by endonuclease restriction with theenzyme XhoI. The PCR product containing the yhfR coding sequence and theXhoI-digested pJ404-LDI were agarose gel-purified using standardlaboratory techniques. The fragments were joined together using theIn-Fusion Advantage PCR Cloning Kit (Clontech Laboratories, Inc.,Mountain View, Calif.), then transformed into chemically competent E.coli GC5 cells (Gene Choice, available from Sigma-Aldrich Co. LLC)following the manufacturer's directions. Transformants were screened,and the proper plasmid was identified through agarose gelelectrophoresis of restriction endonuclease-digested plasmid DNAs. Theproper plasmid was then transformed into electrocompetent E. coli BL21.The resulting plasmid was designated pJ404-LDI.yhfR.

The production of isoprene by BL21 harboring plasmid pJ404-LDI.yhfR maybe assayed as follows. A single colony of BL21 harboring pJ404-LDI.yhfRor pJ404-LDI from LB-agar plates are used to inoculate 10 mL of LB broth(10 g/L yeast extract, 5 g/L Bacto Tryptone, 10 g/L sodium chloride)containing 20 g/L glycerol and 100 μg/ml ampicillin contained in 125 mLErlenmeyer flasks. Flasks are incubated for 16 hours at 37° C. in arotary shaking incubator. After 16 hours, the cultures are diluted usingfresh LB broth containing 20 g/L glycerol and 100 μg/ml ampicillin to anoptical density of 0.16 at 600 nm. 50 ml of the diluted cultures areplaced in 300-mL Erlenmeyer flasks and incubated at 37° C. in a rotaryshaking incubator until the optical density at 600 nm reachesapproximately 0.6, typically 90 minutes. 4 ml of the cultures are thenplaced into 20 ml gas chromatography headspace vials. IPTG (Isopropylβ-D-1-thiogalactopyranoside) is added to 0.1 mM. Cultures are grown for16 hours at 37° C. with shaking.

Isoprene, 3-methyl-2-buten-1-ol and 2-methyl-3-buten-2-ol are measuredas above. The identity of the isoprene peak may be verified using GC/MS,as described above.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, documents,manuscripts and scientific literature cited herein are herebyincorporated by reference.

While the foregoing is directed to embodiments of the present invention,other and further embodiments of the invention may be devised withoutdeparting from the basic scope thereof, and the scope thereof isdetermined by the claims that follow.

What is claimed is:
 1. A non-naturally occurring microbial organismcomprising an isoprene biosynthetic pathway for conversion ofdimethylallyl diphosphate (DMAPP) to isoprene, wherein the isoprenebiosynthetic pathway comprises the conversion of DMAPP to3-methyl-2-buten-1-ol by a 3-methyl-2-buten-1-ol synthase, followed bythe conversion of 3-methyl-2-buten-1-ol to 2-methyl-3-buten-2-ol by a2-methyl-3-buten-2-ol isomerase, followed by the conversion of2-methyl-3-buten-2-ol to isoprene by a 2-methyl-3-buten-2-oldehydratase, wherein the organism comprises an exogenous nucleic acidencoding a 3-methyl-2-buten-1-ol synthase; a 2-methyl-3-buten-2-olisomerase; and a 2-methyl-3-buten-2-ol dehydratase; and wherein theisoprene biosynthetic pathway is expressed at a sufficient level toproduce isoprene.
 2. The non-naturally occurring microbial organism ofclaim 1, wherein the organism overexpresses one or more endogenous orexogenous genes encoding at least one enzyme selected from: an enzyme ofthe methylerythritol phosphate pathway or an enzyme of the mevalonatepathway.
 3. The non-naturally occurring microbial organism of claim 2,wherein the dimethylallyl diphosphate available for conversion toisoprene is increased.
 4. The non-naturally occurring microbial organismof claim 1, wherein the 2-methyl-3-buten-2-ol dehydratase is abi-functional enzyme further comprising 2-methyl-3-buten-2-ol isomeraseactivity.
 5. The non-naturally occurring microbial organism of claim 4,wherein the 2-methyl-3-buten-2-ol dehydratase is a linalooldehydratase-isomerase.
 6. The non-naturally occurring microbial organismof claim 5, wherein the 2-methyl-3-buten-2-ol dehydratase is a linalooldehydratase-isomerase derived from Castellaniella defragrans.
 7. Thenon-naturally occurring microbial organism of claim 1, wherein the3-methyl-2-buten-1-ol synthase is a phosphatase.
 8. The non-naturallyoccurring microbial organism of claim 7, wherein the phosphatase isderived from Bacillus subtilis yqkG, Bacillus subtilis yhfR, orEscherichia coli ytjC.
 9. The non-naturally occurring microbial organismof claim 1, wherein the 3-methyl-2-buten-1-ol synthase is a terpenesynthase.
 10. The non-naturally occurring microbial organism of claim 9,wherein the terpene synthase is a geraniol synthase.
 11. Thenon-naturally occurring microbial organism of claim 10, wherein thegeraniol synthase is derived from Ocimum basilicum, Perilla citriodora,Perilla frutescans, or Cinnamomom tenuipile.
 12. The non-naturallyoccurring microbial organism of claim 9, wherein the terpene synthase isa farnesol synthase.
 13. The non-naturally occurring microbial organismof claim 12, wherein the farnesol synthase is derived from Zea mays orOryza satiza.
 14. The non-naturally occurring microbial organism ofclaim 1, further comprising one or more endogenous or exogenous genesencoding at least one enzyme of the methylerythritol phosphate pathwayselected from: 1-deoxy-D-xylulose-5-phosphate synthase,1-deoxy-D-xylulose-5-phosphate reductoisomerase,4-diphosphocytidyl-2-C-methyl-D-erythritol synthase,4-diphosphocytidyl-2-C-methyl-D-erythritol kinase,2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase,1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase,dimethylallyl-diphosphate/isopentenyl-diphosphate:NAD(P)⁺oxidoreductase,isopentenyl diphosphate isomerase, and combinations thereof.
 15. Thenon-naturally occurring microbial organism of claim 1, furthercomprising one or more endogenous or exogenous genes encoding at leastone enzyme of the mevalonate pathway selected from: acetyl-CoAacetyltransferase, 3-hydroxy-3-methylglutaryl-CoA synthase,3-hydroxy-3-methylglutaryl-CoA reductase, mevalonate kinase,phosphomevalonate kinase, diphosphomevalonate decarboxylase,isopentenyl-diphosphate isomerase and combinations thereof.
 16. Thenon-naturally occurring microbial organism of claim 1, wherein themicrobial organism is selected from bacteria, archaea, eubacteria, yeastand fungi.
 17. The non-naturally occurring microbial organism of claim1, wherein the microbial organism is an Escherichia coli.
 18. Thenon-naturally occurring microbial organism of claim 2, wherein the atleast one enzyme of the methylerythritol phosphate pathway is selectedfrom: 1-deoxy-D-xylulose-5-phosphate synthase,1-deoxy-D-xylulose-5-phosphate reductoisomerase,4-diphosphocytidyl-2-C-methyl-D-erythritol synthase,4-diphosphocytidyl-2-C-methyl-D-erythritol kinase,2-C-methyl-D-erythrito1-2,4-cyclodiphosphate synthase,1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase,dimethylallyl-diphosphate/isopentenyl-diphosphate:NAD(P)⁺oxidoreductase,isopentenyl diphosphate isomerase, and combinations thereof.
 19. Thenon-naturally occurring microbial organism of claim 18, wherein the atleast one enzyme of the methylerythritol phosphate pathway is selectedfrom: 1-deoxy-D-xylulose-5-phosphate synthase,1-deoxy-D-xylulose-5-phosphate reductoisomerase and isopentenyldiphosphate isomerase.
 20. The non-naturally occurring microbialorganism of claim 2, wherein the at least one enzyme of the mevalonatepathway is selected from: acetyl-CoA acetyltransferase,3-hydroxy-3-methylglutaryl-CoA synthase, 3-hydroxy-3-methylglutaryl-CoAreductase, mevalonate kinase, phosphomevalonate kinase,diphosphomevalonate decarboxylase, isopentenyl-diphosphate isomerase andcombinations thereof.
 21. A non-naturally occurring microbial organismof claim 1, wherein the dimethylallyl diphosphate available forconversion to isoprene is increased.
 22. A method of producing isoprene,the method comprising the steps of culturing a non-naturally occurringmicrobial organism of claim 1 in a suitable culture medium containing acarbon source under conditions such that the non-naturally occurringmicroorganism converts at least a part of the carbon source to isoprene,and optionally recovering the isoprene.
 23. The method of claim 22,wherein at least one enzyme of a methylerythritol phosphate pathway orthe mevalonate pathway is overexpressed by the organism.